Development and application of a liquid chromatography coupled to mass spectrometry method for the simultaneous determination of 23 antineoplastic drugs at trace levels.

The goal of this study was to develop a method for the simultaneous quantification of 23 commonly used antineoplastic drugs in a hospital pharmacy, using ultra-high pressure liquid chromatography separation coupled to tandem mass spectrometry detection (UHPLC-MS/MS). The following drugs were investigated: 5-fluorouracil, cytarabine, ganciclovir, gemcitabine, dacarbazine, methotrexate, pemetrexed, busulfan, topotecan, rentitrexed, ifosfamide, cyclophosphamide, etoposide, irinotecan, doxorubicin/epirubicin, vincristine, docetaxel, paclitaxel, daunorubicin, idarubicin, vinblastine, oxaliplatin and carboplatin. The chromatographic separation was performed on a phenyl-hexyl column (2.1 ×100 mm, 1.7 µm) with a gradient elution of methanol and water containing 10 mM ammonium formate adjusted to pH 4.9. All compounds were analyzed in less than 13 min and detected with a triple quadrupole mass spectrometer operating in MRM mode. Limits of detection (LODs) and limits of quantification (LOQs) were comprised between 0.01 and 5 ng.mL-1, and between 0.5 and 5 ng.mL-1, respectively. Accuracies ranged between 117% and 83% at the LOQ, intermediate and upper LOQ concentrations, with relative standard deviations (RSD) inferior to 8%, for all the antineoplastic drugs. Finally, the UHPLC-MS/MS method was successfully applied to the analysis of surface samples to evaluate the chemical contamination by these highly toxic compounds in a chemotherapy preparation unit in a hospital pharmacy with the purpose of monitoring the exposure of health care professionals.

Protein precipitation for the analysis of a drug cocktail in plasma by LC-ESI-MS.

Three protein precipitation (PP) procedures with acetonitrile (ACN), perchloric acid (PA) and trichloroacetic acid (TCA) were investigated for the analysis of a drug cocktail from human plasma samples containing three pharmaceutical compounds and their primary metabolites. For this purpose, a capillary liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) method was developed for the simultaneous analysis of the six tested compounds in less than 6 min. Matrix effect was tested for each PP procedure by means of a post-column infusion system. The three PP techniques were found effective in removing proteins from human plasma and were fully compatible with capillary LC-ESI-MS analysis. However, with acid precipitations, low analyte recovery and a high variability, probably due to analyte coprecipitation, were obtained. Finally, ACN was found to be the most effective PP technique with a recovery higher than 80% and CV inferior to 6%.

Restricted access materials and large particle supports for on-line sample preparation: an attractive approach for biological fluids analysis.

An analytical process generally involves four main steps: (1) sample preparation; (2) analytical separation; (3) detection; and (4) data handling. In the bioanalytical field, sample preparation is often considered as the time-limiting step. Indeed, the extraction techniques commonly used for biological matrices such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are achieved in the off-line mode. In order to perform a high throughput analysis, efforts have been engaged in developing a faster sample purification process. Among different strategies, the introduction of special extraction sorbents, such as the restricted access media (RAM) and large particle supports (LPS), allowing the direct and repetitive injection of complex biological matrices, represents a very attractive approach. Integrated in a liquid chromatography (LC) system, these extraction supports lead to the automation, simplification and speeding up of the sample preparation process. In this paper, RAM and LPS are reviewed and particular attention is given to commercially available supports. Applications of these extraction supports, are presented in single column and column-switching configurations, for the direct analysis of compounds in various biological fluids.

Rapid analysis of fluoxetine and its metabolite in plasma by LC-MS with column-switching approach.

A rapid and sensitive method was developed for the simultaneous determination of fluoxetine and its primary metabolite, norfluoxetine, in plasma. It was based on a column-switching approach with a precolumn packed with large size particles coupled with a liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS). After a simple centrifugation, plasma samples were directly injected onto the precolumn. The endogenous material was excluded thanks to a high flow rate while analytes were retained by hydrophobic interactions. Afterwards, the target compounds were eluted in back flush mode to an octadecyl analytical column and detected by ESI-MS. The overall analysis time per sample, from plasma sample preparation to data acquisition, was achieved in less than 4 min. Method performances were evaluated. The method showed good linearity in the range of 25-1000 ng mL(-1) with a determination coefficient higher than 0.99. Limits of quantification were estimated at 25 ng mL(-1) for fluoxetine and norfluoxetine. Moreover, method precision was better than 6% in the studied concentration range. These results demonstrated that the method could be used to quantify target compounds. Finally, the developed assay proved to be suitable for the simultaneous analysis of fluoxetine and its metabolite in real plasma samples.

Use of large particles support for fast analysis of methadone and its primary metabolite in human plasma by liquid chromatography-mass spectrometry.

A bioanalytical method was developed for the quantitation of methadone (MTD) and its primary metabolite, (EDDP) in plasma. The extraction step was performed within a capillary column packed with large particles (35x0.3 mm I.D.; d(p) 30 micrometer) at high flow-rate conditions (450 microliter/min). The separation was performed on a microbore analytical column (55x2 mm I.D.; d(p) 3 micrometer) coupled to a mass spectrometer (MS). This procedure was based on a column-switching unit. Analytes of interest were retained on the precolumn by hydrophobic interactions and backflushed from the precolumn to the analytical column. The detection was carried out with a MS single quadrupole equipped with an electrospray interface. The total analysis time was 6 min. The limits of quantification were evaluated at 10 and 25 ng/ml for MTD and EDDP, respectively. At this level, good accuracies were obtained for both analytes with repeatability values less than 18%.